Charlotte Volgers and the “Membrane Vesicle Group” at the Department of Medical Microbiology, Maastricht University, focus on membrane vesicles released during infection and inflammation. We interviewed Charlotte about the group’s recent paper “Bead-Based Flow Cytometry For Semi-Quantitative Analysis Of Complex Membrane Vesicle Populations Released By Bacteria And Host Cells”.
What is the background of your research at the Maastricht University?
The “Membrane vesicle group” at the department of Medical Microbiology focuses on membrane vesicles released in the context of infection and inflammation. To this end we study membrane vesicles released by both bacteria and by host cells. Our aim is to establish the involvement of these vesicles in inflammatory processes but also to determine how these vesicles contribute to the development of antibiotic resistance.
Explain the need for accurate particle characterization and quantification in your research area.
In our studies, it is important to determine the membrane vesicle (MV) concentration and size. The formation and characteristics of bacterial MVs are critically determined by the state and the environment of the cell. With respect to Gram-negative bacteria, MVs are formed on the outer membrane. Outer membrane characteristics are particularly sensitive to the nutritional state of the cell and environmental stressors. The qNano is very helpful for the proper characterization of bacterial vesicles released in the context of infection, for example in response to host-associated environmental stressors.
The qNano allows us to determine the accurate MV concentrations which has a two-fold benefit for us:
1) Detailed knowledge on the MV concentrations secreted during infection or different monoculture conditions is an important read-out when studying molecular mechanisms that modulate the MV release.
2) The qNano also allows us to normalize MV concentrations for functional analysis and to match MV isolates from different sources. In this way, we can distinguish whether functional differences are caused by quantitative or compositional differences.
How are you using the qNano Gold to help achieve this?
The MV populations we are interested in are roughly between 50 and 250nm in size. The qNano helps us to analyze these populations and to establish the size distributions of bacterial and host cell-derived vesicles released in the context of infection. Moreover, the qNano also enables us to determine the vesicle concentrations required to assess the physiological activity of these vesicles. Finally, although we have not yet performed such measurements, the qNano also can be used for the determination of the zeta potential of MVs. It might be interesting to investigate if the differential membrane composition of bacterial and host-cell derived vesicles is reflected by differences in the zeta potential.
At present, techniques used for the characterization and quantification are mainly based on microscopy and flow-cytometry. Each of these techniques has its own strengths and limitations. The qNano accurately determines the size and concentration but is unable to discriminate between different MVs in mixed populations of comparable sizes. In our paper on bead-based flow cytometry, we demonstrated that antibody-coated beads can be used for the semi-quantitative determination of specific bacterial and host cell-derived vesicle subpopulations. Moreover, we demonstrated a good correlation between relative (determined by flow cytometry) and absolute (determined by TRPS) numbers indicating that TRPS can provide with a means to convert the relative vesicle concentration to the absolute vesicle concentration. So our flow cytometry based approach is not only highly versatile and accessible, we demonstrate that together with TRPS it can also provide with an approach to establish absolute vesicle concentrations of specific vesicle populations within a heterogeneous vesicle population.
You can read the publication here.