Microbiology

Candida albicans is the pathogenic fungus that most frequently causes infections in humans. It is part of the microbiota commonly found in the skin, gastrointestinal tract, and vaginal mucosa. However, certain conditions, including immunosuppression, excessive use of antibiotics, hormonal changes, the use of medical devices in patients, and individual nutritional status, promote the development of opportunistic infections caused by this fungus. One of the main fungal structures interacting with the host is the cell wall, which is principally composed of chitin, glucan, and proteins. The cell wall plays key functions for the cell, such as osmotic protection; it is also responsible for cellular shape and acts as a signaling hub in response to environmental changes. Cell wall proteins participate in diverse cellular functions, such as attachment to surfaces and cell wall structure; some possess catalytic or transport activities. In this protocol, we show the methodology for isolating cell wall proteins covalently linked or not to cell wall components that can be previously labeled with [¹⁴C]-L-lysine by the action of the fungal transglutaminase localized in the cell wall. We use an extraction method by mechanical cell disruption and washing with 2 M NaCl, whose ionic strength eliminates contaminating proteins from other organelles, through subsequent serial treatments with SDS, chitinase, and zymolyase.

The ribosome, a complex macromolecular machine, plays a vital role in cellular translation. To investigate its structure and conduct in vitro experiments, isolating the ribosomes from cells is the first step. While isolating ribosomes from bacterial cells is routine, obtaining them from mycobacteria proves challenging due to the protective mycolic acid layer, which hinders cell lysis. In this study, we present a straightforward and efficient protocol for isolating ribosomes from Mycobacterium smegmatis. Additionally, we introduce a co-sedimentation assay using density gradient ultracentrifugation, providing a simple yet powerful method for studying ribosome–protein interactions. The re-association assay also offers a practical approach for obtaining tRNA-free 70S ribosomes and evaluating the anti-association properties of potential ligands. While these assays are commonly used, our protocol stands out for its simplicity, requiring limited specialized instruments. These methods can also be scaled up or down per requirement. By employing sonication for cell rupture and utilizing basic lab equipment for ultracentrifugation-based assays, our method greatly simplifies ribosome isolation and related research.

The bacterial membrane vesicles (MVs) are non-replicative, nanoscale structures that carry specific cargos and play multiple roles in microbe–host interactions. An appropriate MV isolation method that mimics complex pathogen infections in vivo is needed. After bacterial MVs extraction, flagella or pili can be frequently observed along with MVs by transmission electron microscope (TEM). Recently, MVs from Pseudomonas aeruginosa were found to coexist with Pf4 phages, and this MV–phages complex exhibited a different impact on host cell innate immunity compared with MVs or phages solely. The presence of this MVs–phages complex simulates the real condition of complex pathogen infections within the host. This protocol outlines the extraction of the MVs and Pf4 phages complex of P. aeruginosa PAO1, including the respective isolation and qualification approaches. Our step-by-step bacterial MVs–phages complex extraction protocol provides valuable insights for further studying microbe–host cell interactions and the development of novel phage therapies.

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.

Throughout their life cycle, bacteria shed portions of their outermost membrane comprised of proteins, lipids, and a diversity of other biomolecules. These biological nanoparticles have been shown to have a range of highly diverse biological activities, including pathogenesis, community regulation, and cellular defense (among others). In recent publications, we have isolated and characterized membrane vesicles (MVs) from several species of Lactobacilli, microbes classified as commensals within the human gut microbiome (Dean et al., 2019 and 2020). With increasing scientific understanding of host-microbe interactions, the gut-brain axis, and tailored probiotics for therapeutic or performance increasing applications, the protocols described herein will be useful to researchers developing new strategies for gut community engineering or the targeted delivery of bio-active molecules.

Graphic abstract:

Figure 1. Atomic force microscopic image of Lactobacillus casei ATCC 393 bacteria margins (white arrows) and membrane vesicles (black arrows)

We have adapted a previous procedure and improved an approach that we named yChEFs (yeast Chromatin Enriched Fractions) for purifying chromatin fractions. This methodology allows the easy, reproducible and scalable recovery of proteins associated with chromatin. By using yChEFs, we bypass subcellular fractionation requirements involved when using zymolyase to obtain the spheroplast, which is employed in many other procedures. Employing small amount of culture cells and small volumes of solutions during the yChEFs procedure is very useful to allow many samples to be handled at the same time, and also reduces costs and efforts. The purified proteins associated with chromatin fractions obtained by yChEFs can be analyzed by Western blot (Figure 1) or combined with mass spectrometry for proteomic analyses.

Gram-negative bacteria naturally release outer membrane vesicles (OMVs) to the surrounding environment. OMVs contribute to multiple processes, such as cell-cell communication, delivery of enzymes and toxins, resistance to environmental stresses and pathogenesis. Little is known about OMVs produced by plant-pathogenic bacteria, and their interactions with host plants. The protocol described below discusses the isolation process of OMVs from Xanthomonas campestris pv. campestris strain 33913, a bacterial pathogen of Crucifiers. Nevertheless, this protocol can be used and/or adapted for isolation of OMVs from other phytopathogenic bacteria to promote the study of OMVs in the context of plant-microbe interactions.

Outer membrane vesicles (OMVs) represent a unique sub-cellular compartment of bacteria that may act as a scaffold for various extracellular activities, including intercellular signaling. Myxococcus xanthus (M. xanthus) is a predatory bacterium that engages in cell-cell behaviors such as fruiting body formation and contact dependent lysis of other microbes. The OMVs of M. xanthus have been shown to have an elaborate architecture of chains and tubes that can connect cells within a biofilm. These higher order OMV structures have been shown to contain proteins exchanged for community behaviors and small molecules that have antibiotic activities, and may help facilitate directed exchange. M. xanthus OMVs allow material transfer between neighboring cells for motility and predation.

The gram-negative curved bacillus Vibrio cholerae (V. cholerae) causes the severe diarrheal illness cholera. The work presented here is to assess whether unsaturated fatty acids (UFAs), such as linoleic acid, have the potential to directly affect proteins involved in DNA binding because they are able to enter the cell. In this protocol, we show how to measure linoleic acid entering V. cholerae when added exogenously and determine whether it is able to enter the cytoplasm. This protocol will quantify how much linoleic acid is able to enter the cell and then identify the amount of linoleic acid that stays in the membrane or ultimately enters the cytoplasm.

Many postitive-stranded RNA viruses, such as Hepatitis C virus (HCV), highjack cellular membranes, including the Golgi, ER, mitchondria, lipid droplets, and utilize them for replication of their RNA genome or assembly of new virions. By investigating how viral proteins associate with cellular membranes we will better understand the roles of cellular membranes in the viral life cycle. Our lab has focused specifically on the role of lipid droplets and lipid-rich membranes in the life cycle of HCV. To analyze the role of lipid-rich membranes in HCV RNA replication, we utilized a membrane flotation assay based on an 10-20-30% iodixanol density gradient developed by Yeaman et al. (2001). This gradient results in a linear increase in density over almost the entire length of the gradient, and membrane particles are separated in the gradient based on their buoyant characteristics. To preserve membranes in the lysate, cells are broken mechanically in a buffer lacking detergent. The cell lysate is loaded on the bottom of the gradient, overlaid with the gradient, and membranes float up as the iodixanol gradient self-generates. The lipid content of membranes and the concentration of associated proteins will determine the separation of different membranes within the gradient. After centrifugation, fractions can be sampled from the top of the gradient and analyzed using standard SDS-PAGE and western blot analysis for proteins of interest.