微生物学

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.

The flagellum is required for bacterial swimming and swarming motility. In the biphasic Salmonella enterica serovar Typhimurium (S. Typhimurium), the flagellar filament is build up by two distinct monomeric subunits, flagellin FliC and FljB. S. Typhimurium has the ability to switch between two flagellins, FliC and FljB, in a phase-variable manner. The switch to FliC is called phase H1 and considered important for bacterial growth and survival in the spleen in a murine infection model of typhoid fever. Flagellin is secreted as monomeric subunits, but the majority of flagellin is polymerized upon secretion as the flagellar filament. Salmonella flagellin has traditionally been isolated through a process involving multiple steps of centrifugation and acid treatment. Here, we delineate a simplified protocol for preparing Salmonella´s flagellin for analytical purpose to determine the amount of flagellin without the aid of antibodies. The growth conditions used were stationary phase, logarithmic phase and a low oxygen and high salt condition mimicking the gastrointestinal tract. Flagellin expression of other source organisms, such as other serovars of Salmonella enterica and Escherichia coli, including flagellar phase- or genetic variants can be analysed. Flagellin expression analysis complements flagella-associated phenotype analysis such as swimming and swarming behaviour.

Recently, membrane vesicle (MV) production was described in Gram-positive bacteria, which harbor a variety of components such as toxins, antibiotic resistance proteins, proteases, DNA, and immune modulators. Free lipids have the ability to form micelles, thus it is important to rule out spontaneous association of lipids into vesicle-like structures and rather, that MVs are produced naturally by a metabolically active cell. Here, we describe a protocol utilizing the polysaccharide, glucuronoxylomannan (GXM) from Cryptococcus neoformans (C. neoformans) as a marker to differentiate naturally produced MVs from vesicles that form spontaneously in the Gram-positive model organism, Bacillus subtilis (B. subtilis). MVs are purified from bacterial cultures grown in the presence of GXM; MVs naturally produced by cells would not contain GXM in the lumen whereas vesicular structures forming in the media could encapsulate GXM and this can be visualized via immunogold transmission electron microscopy.

Bacterial biofilms are associated clinically with many bacterial infections including those caused by bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. In recent years, extracellular vesicles produced by bacteria have been isolated from biofilm communities. Vesicles have been described in depth and can encapsulate various virulence factors including toxins and immunomodulatory compounds. Vesicles may be important for virulence and survival by serving as a vehicle for the secretion and concentrated delivery of these molecules. Studying extracellular vesicles is an important step towards understanding biofilm formation, structure, and disruption with the ultimate goal of preventing or treating hospital infections caused by bacterial pathogens residing in biofilms. Here we describe the protocol for isolating vesicles from biofilm produced by Bacillus subtilis.

Protein fractionation is a useful separation process which divides membrane proteins (including those located in the outer and inner membrane) and cytoplasmic proteins into discrete fractions. Fractionation of proteins can simplify analysis of the numbers of proteins present, and therefore make easier to characterize any environmentally or mutation induced changes in expression profiles, or changes in protein strucutre resulting from post-translational modification. This protocol is derived from Haigh et al. (2013) and it is specific to Gram negative bacteria.