Nobel Prize STORM technology for meningococcal research

In recent years, the international rise of ultra-high-resolution microscopy imaging technology has provided scientists with the latest imaging methods, enabling scientists to obtain more details of bacteria and viruses, and providing more support for the development of the field of biomedicine.
This article will share some of the advances made by the 2014 Nobel Prize in Chemistry STORM super-resolution microscopy in the study of meningococcal infection mechanisms. Teachers are welcome to exchange and discuss.

Meningococcus is a gram-negative bacterium that causes epidemic meningitis and sepsis worldwide, the authors first studied the mobility of CD147 in meningococcal infection through single-molecule tracking experiments, and then investigated the role of two sphingolipids GM1 and Gb3 in the process of meningococcal infection using fluorescently labeled STxB and CTxB subunits, respectively, by super-resolution microscopy observation by structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM), GM1 aggregation around meningococci was shown, highlighting its importance for bacterial invasion.

In healthy individuals, meningococci may exist as symbionts in the nasopharynx without affecting the host, and in some cases, bacteria can enter the bloodstream and adhere to the endothelial cells of the blood microvessels, leading to the inflammatory process and disruption of the blood-cerebrospinal fluid barrier, and in severe cases can cause sepsis and progressive fatal shock, which is key to the development of the disease into meningitis.

Recently, super-resolution microscopy by direct stochastic optical reconstruction microscopy (dSTORM) has shown that binding of meningococci to endothelial cells requires CD147/β2-adrenoceptor aggregation at bacterial adhesion sites, and local enrichment of CD147/β2-adrenergic receptor complexes in the plasma membrane may allow bacteria to adhere to the vessel wall in vivo and resist hemodynamics, since receptor accumulation at bacterial adhesion sites requires high mobility in the plasma membrane, The authors performed live-cell single-molecule tracking experiments of CD147 using an N-terminus-bound monoclonal antibody (MEM-6/1) bound to the photostable fluorescent dye SeTau-647.

Figure 1. Single molecule tracking of CD147 on HBMEC by monoclonal antibodies

Sphingolipids are usually important host cell targets for a large number of pathogens, meningococcal fimbriae depend on ganglioside binding, there are two well-studied glycolipids with receptor function are monosialic acid tetrahexosylganglioside GM1 and bulbar triglycosylceramide Gb3, in order to study possible changes in lipid tissue during meningococcal adhesion, The authors used cholera toxin B (CTxB) and Shiga toxin B (STxB) subunits to observe the distribution of two glycosphingolipids GM1 and Gb3 in the plasma membrane of cerebral endothelial cells, respectively.

The experimental results showed that after infection with meningococci, the plasma membrane distribution of Gb3 remained unchanged, on the contrary, GM1 showed a significant increase in fluorescence intensity around adhesive meningococci on the HBMEC plasma membrane, and the data clearly demonstrated the strong aggregation of gangliosides GM1 around adhering bacteria, while uninfected HBMEC showed a uniform distribution of GM1 in the plasma membrane.

Figure 2. Fluorescence imaging of Gb3 in the plasma membrane of brain endothelial cells during meningococcal infection (left), fluorescence imaging of GM1 in HBMEC after infection with meningococci expressing GFP (right)

To rule out non-specific binding of CTxB and STxB to meningococci, the authors inoculated the bacteria on HBMEC-free glass, labeled and imaged by dSTORM, and the corresponding images showed that the two sphingolipids did not bind nonspecifically to meningococci.

Figure 3. dSTORM images of GM1 and Gb3 of GFP-expressing meningococci (green), without HBMEC labeled with Alexa Fluor 647 conjugated CTxB or STxB.

In summary, glycosphingolipids are important pathogen receptors, and blocking GM1 can significantly reduce infection efficiency, which means that plasma membrane gangliosides are important for bacterial invasion. Therefore, the increase in invasion potency should be more pronounced in the presence of gangliosides in human epithelial cells interacting with fimbriae, and the authors suggest that this mechanism may play an important role in the initial uptake from the nasopharynx to the bloodstream, and blocking this interaction may be a promising way to avoid meningococcal transmission and threaten life-threatening and help develop treatments for bacterial clearance.
In this paper, we mainly use STORM technology to mark and track molecular position relationships. This discovery of the 2014 Nobel Prize in Chemistry has been industrialized in China. The ultra-high-resolution microscopic imaging system iSTORM released by Ningbo Lixian Intelligent Technology Co., Ltd. (INVIEW) has been highly recognized by more than 50 scientific research groups and more than 100 researchers for its excellent characteristics such as nanoscale observation accuracy, high stability, wide environmental applicability, rapid imaging, and simple operation, using 3D random optical reconstruction technology, high-precision cell real-time locking technology, and multi-channel simultaneous imaging technology.