Mapping the associations between Wolbachia and microtubules in filarial worms

The National Museum of Natural History (MNHN) is a public research and higher education institution dedicated to the study of biodiversity, evolution and the interactions between organisms and their environment. Within the MNHN, the MCAM unit (Molecules of Communication and Adaptation in Microorganisms) focuses on the molecular and physiological mechanisms involved in the communication and adaptation of microorganisms. The PPL team (Free-Living Protists and Parasites), which forms part of this unit, studies the diversity, biology and ecology of protists and parasites.

Current state of the art and background

Filariae are parasitic nematodes responsible for major human diseases. They harbour endosymbiotic bacteria of the genus Wolbachia, which are essential for their development, survival and reproduction. The maternal transmission of these bacteria is a key stage in the parasitic life cycle, one that is still poorly understood at the cellular level. During the parasite’s development, the bacterium changes location and gradually colonises the reproductive tissues, first the oocytes and then the embryos. Whilst this process is relatively well described at the tissue level, the cellular mechanisms enabling Wolbachia to reach the correct compartments at the right time remain largely unknown.

In insects, Wolbachia utilises the host’s cytoskeleton, in particular microtubules and molecular motors, to facilitate its intracellular transport. In filarial worms, these mechanisms remain to be elucidated. Preliminary results obtained by the team suggest the existence of post-translational modifications of tubulin, which may give rise to different populations of microtubules with distinct properties.

Objective

This M2 research project aims to map the interactions between Wolbachia and microtubules in filarial worms. Studying the interactions between Wolbachia and microtubules requires a resolution higher than that of confocal microscopy, as it involves structures of very different sizes: a filariae reproductive tract spanning several centimetres, bacteria measuring around 1 µm, and microtubules with a diameter of 25 nm. These interactions therefore occur well below the resolution of confocal microscopy; however, thanks to their well-defined geometry and the variety of available markers, microtubules provide a reference system particularly well-suited to super-resolution approaches.

Proposed actions and experimental approach.

The student will first isolate the filariae, dissect the reproductive tissues and prepare the samples for analysis by expansion microscopy (ExM), a technique that physically enlarges biological structures whilst preserving their organisation, thereby achieving nanometre-scale resolution using conventional optical microscopes. Combined labelling techniques will be employed, combining Wolbachia detection (FISH) with the analysis of different types of tubulin using several antibodies directed against total tubulin and its post-translational modifications. This approach will enable the simultaneous visualisation of microtubules and bacteria at a subcellular level sufficient to map their interactions in detail.

This internship will enable the acquisition of skills in the preparation of complex samples, advanced microscopy (including ExM) and image analysis, whilst contributing to a better understanding of the cellular mechanisms involved in Wolbachia transmission.

Expected results and future prospects

This approach will enable the simultaneous visualisation of microtubules and bacteria at a subcellular level sufficient to map their interactions in detail. It will allow us to select candidates for future functional studies (RNA interference, pharmacological disruption of the cytoskeleton, in conjunction with our work on oxfendazole, which profoundly disrupts the organisation of microtubules).

First-year Master’s degree in cell biology or parasitology, or One Health
An interest in microscopy and experimental work
Experience in immunofluorescence or microscopy would be an advantage
Attention to detail, the ability to work independently and as part of a team