Watching an infection unfold with a sphingolipid probe

When a class of small molecules does not metabolize properly in the human body, there may be some large consequences. These molecules, called sphingolipids, are located in the plasma membrane of cells and play critical functions in cellular structure and cell signaling (1). When a specific type of sphingolipid, known as sphingomyelin, is left unchecked due to a genetic mutation, sphingomyelin illnesses can manifest, such as Niemann-Pick disease or Parkinson’s disease (2,3). Scientists have also identified sphingomyelins as important in the pathogenesis of certain infections, most notably COVID-19 (4).

Even with growing awareness of their presence in many diseases, sphingomyelin and their counterpart enzymes, sphingomyelinases, are quite understudied, said Jürgen Seibel, an organic chemist at the University of Würzburg. “With small molecules, it is always very difficult to visualize where they are, how much there is, and which enzymes are surrounding those molecules,” he added.

Due to their size and location in the cell membrane, imaging sphingomyelin requires super-resolution techniques that are often complex and expensive. So, Seibel and an interdisciplinary research team designed a new, high-resolution visualization method by synthesizing modified sphingomyelin called trifunctional sphingomyelin (TFSM) (5). They monitored the synthesized lipid’s metabolism during Chlamydia trachomatis infiltration of human cells using expansion microscopy, an economical laboratory technique, to get a nanoscale glimpse of the cell environment. Their work was published in Nature Communications.

Seibel and his team developed TFSM using “click chemistry” to piece together three functional groups on to a natural form of sphingomyelin. The TFSMs had two fluorescent molecular probes attached to each end of the sphingomyelin that can detect molecular proximities by turning on and off as energy transfers within the TFSM. The last functional group was a primary amino group that anchors the TFSM to a hydrogel while the sample physically expands during fluorescence imaging for the expansion microscopy process.

The researchers demonstrated this new technique by observing bacterial sphingomyelinase activity from C. trachomatis, an obligate intracellular pathogen primarily responsible for one of the leading sexually transmitted infections in the United States (6). The bacteria harness the host’s sphingomyelin to develop inclusions, which are bubbles inside the human cell that the pathogen uses to replicate. Chlamydia species also have two main developmental forms — mature reticulate bodies and infectious elementary bodies.

The team discovered that the pathogen could break down TFSM similarly to the natural human sphingomyelin, and the products primarily congregated in the bacterial inclusions. Using expansion microscopy, the researchers also found that the TFSM metabolites were more abundant in the elementary bodies, confirming the unique process of infection for this pathogen.

“It is a complete interplay of different molecules, and there seems to be a lot going on in the cell and on the cell,” Seibel said. “In this study, we have this great achievement by observing the next generation of molecules that behave like natural molecules and tell us the conditions of infections.”

From these findings of the bacterial sphingomyelinase reactions in human cells, other researchers see the potential of this TFSM visualization technique expanded to sphingolipid interactions in general. 

“If we can extrapolate this kind of reaction to other molecular species, this technique would become extremely useful,” said Ruth Gordillo, an analytical chemist at the University of Texas Southwestern Medical Center, who was not involved in this study. “For sphingomyelins, this is one of the best techniques I have seen at the level where you can actually see molecular localization at the cellular level in the membrane.”

Gordillo works closely with techniques like mass spectrometry imaging to determine and quantify sphingolipid metabolites for a range of diseases, such as liver disease and diabetes. In this line of work, the visualization resolution or cost can be major hurdles. 

She hopes through this new method of visualization, monitoring sphingolipid dynamics may become the norm for better understanding infections as well as a variety of other diseases.

References

1. Quinville, B. et al. A Comprehensive Review: Sphingolipid Metabolism and Implications of Disruption in Sphingolipid Homeostasis. Int J Mol Sci  22, 5793 (2021).
2. Tirelli, C. et al. The Genetic Basis, Lung Involvement, and Therapeutic Options in Niemann–Pick Disease: A Comprehensive Review. Biomolecules  14, 211 (2024).
3. Alessenko, A.V. & Albi, E. Exploring Sphingolipid Implications in Neurodegeneration. Front Neurol  11, 437 (2020).
4. Toro, D.M. et al. Plasma sphingomyelin disturbances: Unveiling its dual role as a crucial immunopathological factor and a severity prognostic biomarking in COVID-19. Cells  12, 1938 (2023).
5. Rühling, M. et al. Trifunctional sphingomyelin derivatives enable nanoscale resolution of sphingomyelin turnover in physiological and infection processes via expansion microscopy. Nat Commun  15, 7456 (2024).
6. Vermund, S.H. et al. Sexually Transmitted Infections: Adopting a Sexual Health Paradigm. National Academies of Sciences, Engineering, and Medicine, Washington, DC: The National Academies Press (2021).