Microbiology

The study of carbohydrate–protein interactions is crucial for clarifying biological processes and identifying potential drug candidates. However, due to the complex structure of carbohydrates, such as high molecular weight, dynamic flexibility, and high solution viscosity, it is challenging to study their interactions with diverse proteins. Conventional analytical techniques like isothermal titration calorimetry (ITC), X-ray crystallography, molecular dynamics (MD) simulations, and nuclear magnetic resonance (NMR) spectroscopy have limitations in revealing these molecular interactions. Surface plasmon resonance (SPR), an advanced optical biosensor technique, overcomes these limitations. It enables real-time, label-free monitoring of the interaction dynamics between carbohydrates and proteins through a continuous flow over a chip surface. In this study, we utilized SPR-based techniques to explore the interaction of capsular polysaccharides (CPS) of Klebsiella pneumoniae and the enzyme KpACE (K. pneumoniae acetylated capsule esterase). Our SPR-based analytical platform has several advantages, including shorter experimental time, a simulated physiological state, and minimal sample requirements for investigating carbohydrate–protein interactions. This approach expands the applicability scope of SPR technology and provides a valuable tool for a wide range of research. By using SPR, we successfully verified that KpACE acts on the acetyl groups of CPS, demonstrating its enzymatic activity, which is crucial for understanding the pathogenic mechanism of K. pneumoniae and developing potential antibacterial drugs.

Carbohydrates serve crucial functions in most living cells, encompassing structural and metabolic roles. Within the realms of plant and algal biology, carbohydrate biosynthesis and partitioning play pivotal roles in growth, development, stress physiology, and various practical applications. These applications span diverse fields, including the food and feed industry, bioenergetics (biofuels), and environmental management. However, existing methods for carbohydrate determination tend to be costly and time-intensive. In response to that, we propose a novel approach to assess carbohydrate partitioning from small samples. This method leverages the differential solubility of various fractions, including soluble sugars, starch, and structural polymers (such as cellulose). After fractionation, a straightforward spectrophotometric analysis allows for the quantification of sugars.

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

Dolichol diphosphate-linked oligosaccharides (LLO) are the sugar donors in N-glycosylation, a fundamental protein post-translational modification of the eukaryotic secretory pathway. Defects in LLO biosynthesis produce human Congenital Disorders of Glycosylation Type I. The synthesis of LLOs and the transfer reactions to their protein acceptors is highly conserved among animal, plant, and fungi kingdoms, making the fission yeast Schizosaccharomyces pombe a suitable model to study these processes. Here, we present a protocol to determine the LLO patterns produced in vivo by S. pombe cells that may be easily adapted to other cell types. First, exponentially growing cultures are labeled with a pulse of [¹⁴C]-glucose. LLOs are then purified by successive extractions with organic solvents, and glycans are separated from the lipid moieties in mild acid hydrolysis and a new solvent extraction. The purified glycans are then run on paper chromatography. We use a deconvolution process to adjust the profile obtained to the minimal number of Gaussian functions needed to fit the data and determine the proportion of each species with respect to total glycan species present in the cell. The method we provide here might be used without any expensive or specialized equipment. The deconvolution process described here might also be useful to analyze species in non-completely resolved chromatograms.

Graphical abstract:

Workflow for the labeling, extraction, separation, and identification of LLO species in S. pombe. (A) Radioactive pulse of S. pombe cells with [¹⁴C]-glucose for 15 min at 28 °C. (B) Organic extraction of LLOs from labeled yeasts sequentially using methanol, chloroform, H₂O, chloroform:methanol:H₂O (1:1:0.3), 0.02 M HCl (to separate glycans from dolichol), and chloroform:methanol:H₂O (1:16:16). (C) Preparation of the sample for chromatography on paper: drying by airflow and radioactivity check. (D) Loading of samples in chromatographic paper and descendent chromatography in a glass chamber. The obtained plots (CPM versus running distance) need to be analyzed to identify single glycan species.

Lipopolysaccharides (LPS) (or lipooligosaccharides [LOS], which lack the O-antigen side chains characteristic of LPS), and outer membrane proteins (OMP) are major cell-surface molecules in the outer membrane (OM) of gram-negative bacteria. The LPS is responsible for causing endotoxic shock in infected hosts and, in conjunction with some OMPs, provides protection to the bacterium against host innate immune defenses and attachment to host cells. Electrophoretic analysis can provide valuable information regarding the size, number, and variability of LPS/LOS and OMP components between bacterial strains and mutants, which aids in understanding the basic biology and virulence factors of a particular species. Furthermore, highly purified extracts are normally not required if only electrophoretic analysis is to be done, and various methods have been established for such procedures. Here, we review ameliorated procedures for fast and convenient extraction of LPS/LOS and protein-enriched outer membranes (PEOM) for optimal electrophoretic resolution. Specifically, we will describe the phenol-based micro-method for LPS/LOS extraction, a differential extraction procedure with sodium lauryl sarcosinate for PEOM, and gel preparation for electrophoretic analysis of LPS/LOS samples in detail.

Graphic abstract:

Workflow for the preparation and analysis of LPS/LOS and PEOM.