Shedding New Light on Bacterial Outer Membrane Vesicles
Written by: Aarshi Singh, Wittenberg Lab
Imagine that you want to convey an urgent message to your friend about an upcoming storm. There are several ways this can be done: through the use of voice, text, or simple body language. However, opportunities for connection are limited for less complex creatures like bacteria, fungi, parasites, etc. Bacteria and other simpler organisms communicate about antibiotics or infections using extracellular vesicles.
Figure 1: Unlocking the Secrets of Extracellular Vesicles: Nature’s Messengers Journeying Through the Body. (Created with Biorender.com)
Extracellular vesicles (EVs) are small membrane-enclosed structures that originate from the membrane of bacteria, cells, or similar structures1. These vesicles are released into the extracellular space and play a vital role in intercellular communication. They carry various biomarkers and genetic material, enabling the organisms to exchange information. For instance, when a bacterium experiences stress and produces a specific protein, that protein is packaged within these vesicles and transmitted to neighboring bacteria, signaling them to initiate production of the same protein.
In the realm of EVs, a remarkable diversity exists. The composition and characteristics of EVs can vary significantly due to the diverse cellular origins and physiological states of the parent cells. This results in unique cargo content and surface properties of the vesicles. For example, different cell types give rise to EVs with distinct subsets of proteins, nucleic acids, lipids, or metabolites, reflecting their specific functions within the body. However, this post will specifically focus on EVs derived from gram-negative bacteria.
While both gram-negative and gram-positive bacteria release vesicles, gram-negative bacteria possess a unique structure characterized by an inner and outer membrane, with the latter enabling the direct secretion of outer membrane vesicles (OMVs). These OMVs from gram-negative bacteria will be the primary subject of exploration in this blog2.
While bacterial vesicles have been recognized for their role in intercellular communication, it is important to note that they can also contribute to the pathogenesis of bacterial infections by serving as vehicles for spreading virulence factors throughout the body3. Interestingly, their genetically modified counterparts have shown several pharmaceutical applications such as FDA approved meningitis vaccine4 and targeted drug delivery for cancer treatment5.
Despite the promising potential of OMVs in the pharmaceutical industry, their full utilization has not been realized yet. One of the key challenges that hinders their widespread application is the inherent heterogeneity observed among OMVs. It is fascinating to note that vesicles, even when isolated from the same population of bacteria at the same time, can exhibit several heterogeneities including differences in size and protein content6,7. Due to the heterogeneities observed in these vesicles, they have been reported to exert diverse physiological effects, such as the entry method in the cell and the presence of certain surface proteins. Understanding and studying these heterogeneities is particularly important before considering the utilization of OMVs for pharmaceutical purposes.
Figure 2: Bacteria releasees heterogenous population of OMVs. (Created with Biorender.com)
Despite the importance, detecting vesicle heterogeneities can be difficult and expensive. Traditional assay analyzes the vesicles population as a whole, which can mask any single vesicle’s heterogeneity. Furthermore, if you are interested in understanding size based heterogeneity, it requires separating the vesicles population based on size, using methods such as density centrifugation or size exclusion chromatography, which adds to the cost of the method.
While there are several single vesicle analysis methods, such as flow cytometry, they also exhibit drawbacks. In flow cytometry, the continuous movement of particles in a fluid stream poses challenges in retracing and analyzing any observed heterogeneities in detail. An alternative approach to analyze single particle heterogeneity is electron microscopy. However, it is important to note that electron microscopy can be invasive for biological samples and has the potential to cause damage to them. Additionally, both flow cytometry and electron microscopy are costly techniques that require specialized equipment and expertise.
Given the significant role of bacterial vesicles in vaccine development and therapeutic applications, it becomes imperative to conduct further research to comprehensively understand their heterogeneity. By gaining a deeper understanding of the diverse characteristics and functional variations within OMVs, we can unlock their full potential and optimize their utilization in the field of immunization and therapeutics.
To overcome this limitation, Singh et al8 recently reported a novel multiple parameter analysis method using a general-purpose fluorescence microscope to determine the size of vesicles and unravel any size-based heterogeneities. This innovative approach offers a promising solution for studying individual vesicles and their heterogeneities, without the need for expensive and invasive methods. This method is particularly innovative as it eliminates the need for specialized instrumentation, making it more accessible for researchers. It also offers the advantage of facilitating further analysis when specific heterogeneities are identified. Additionally, this method can be optimized and tailored to meet specific research needs, allowing for customization and versatility in studying OMV heterogeneity.
Our novel approach utilizing single-particle fluorescence sizing has proven to be an effective method for studying individual vesicles and their heterogeneity. Furthermore, our results have important implications for understanding the functionality of vesicles and their interaction with target cells. By elucidating the diverse characteristics of these vesicles, we gain valuable insights into their functionality and their interactions with target cells, paving the way for potential advancements in therapeutic interventions and a deeper understanding of microbial communication networks.
Deciphering the messages encoded within bacterial vesicles provides us with a profound understanding of how bacteria communicate across the vast landscape of the human body, much like the text message you sent to your friend about the approaching storm.
References:
(1) Simeone, P.; Bologna, G.; Lanuti, P.; Pierdomenico, L.; Guagnano, M. T.; Pieragostino, D.; Del Boccio, P.; Vergara, D.; Marchisio, M.; Miscia, S.; Mariani-Costantini, R. Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers. Int. J. Mol. Sci. 2020, 21 (7), 2514. https://doi.org/10.3390/ijms21072514.
(2) Jan, A. T. Outer Membrane Vesicles (OMVs) of Gram-Negative Bacteria: A Perspective Update. Front. Microbiol. 2017, 8, 1053. https://doi.org/10.3389/fmicb.2017.01053.
(3) Balsalobre, C.; Silvan, J. M.; Berglund, S.; Mizunoe, Y.; Uhlin, B. E.; Wai, S. N. Release of the Type I Secreted Alpha-Haemolysin via Outer Membrane Vesicles from Escherichia Coli. Mol. Microbiol. 2006, 59 (1), 99–112. https://doi.org/10.1111/j.1365-2958.2005.04938.x.
(4) Uli, L.; Castellanos-Serra, L.; Betancourt, L.; Domínguez, F.; Barberá, R.; Sotolongo, F.; Guillén, G.; Pajón Feyt, R. Outer Membrane Vesicles of the VA-MENGOC-BC® Vaccine against Serogroup Bof Neisseria Meningitidis: Analysis of Protein Components by Two-Dimensional Gel Electrophoresis and Mass Spectrometry. PROTEOMICS 2006, 6 (11), 3389–3399. https://doi.org/10.1002/pmic.200500502.
(5) Kim, O. Y.; Park, H. T.; Dinh, N. T. H.; Choi, S. J.; Lee, J.; Kim, J. H.; Lee, S.-W.; Gho, Y. S. Bacterial Outer Membrane Vesicles Suppress Tumor by Interferon-γ-Mediated Antitumor Response. Nat. Commun. 2017, 8 (1), 626. https://doi.org/10.1038/s41467-017-00729-8.
(6) Turner, L.; Bitto, N. J.; Steer, D. L.; Lo, C.; D’Costa, K.; Ramm, G.; Shambrook, M.; Hill, A. F.; Ferrero, R. L.; Kaparakis-Liaskos, M. Helicobacter Pylori Outer Membrane Vesicle Size Determines Their Mechanisms of Host Cell Entry and Protein Content. Front. Immunol. 2018, 9, 1466. https://doi.org/10.3389/fimmu.2018.01466.
(7) Gardiner, C.; Shaw, M.; Hole, P.; Smith, J.; Tannetta, D.; Redman, C. W.; Sargent, I. L. Measurement of Refractive Index by Nanoparticle Tracking Analysis Reveals Heterogeneity in Extracellular Vesicles. J. Extracell. Vesicles 2014, 3 (1), 25361. https://doi.org/10.3402/jev.v3.25361.
(8) Singh, A. N.; Nice, J. B.; Brown, A. C.; Wittenberg, N. J. Identifying Size-Dependent Toxin Sorting in Bacterial Outer Membrane Vesicles; preprint; Biophysics, 2023. https://doi.org/10.1101/2023.05.03.539273.
