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Updated: Jun 24, 2025
Investigating the Phagocytosis of Leishmania using Confocal Microscopy
Published on: July 29, 2021
A Multi-Color Immunofluorescence Assay to Distinguish Intracellular From External Leishmania Parasites
Arani Datta1, Umaru Barrie1,2, Dawn M Wetzel1,3
1Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.
Insights
This study details a protocol for infecting macrophages with Leishmania parasites to understand host-pathogen interactions. The method quantifies intracellular parasites and aids in developing new leishmaniasis treatments.
Area of Science:
- Parasitology
- Immunology
- Cell Biology
Background:
- Leishmaniasis is a neglected tropical disease caused by intracellular protozoan parasites of the genus Leishmania.
- Limited therapeutic options and parasite immune evasion necessitate novel treatment strategies.
- Understanding Leishmania internalization by host cells is crucial for developing new interventions.
Purpose of the Study:
- To characterize the host cell signaling pathways involved in Leishmania internalization.
- To provide a detailed protocol for infecting mammalian macrophages with Leishmania parasites.
- To enable quantification of intracellular parasites and host cells for studying host-pathogen dynamics.
Main Methods:
- Infection of mammalian macrophages with Leishmania promastigote or amastigote forms.
- Utilizing immunofluorescence microscopy for visualization of parasites and host cells.
- Employing semi-automated analysis for quantifying intracellular and extracellular parasites and macrophages.
Main Results:
- The protocol successfully visualizes and quantifies multiple intracellular forms of Leishmania within macrophages.
- The method allows for detailed study of host-pathogen interactions during Leishmania infection.
- The protocol is adaptable for researchers to modify based on specific experimental needs.
Conclusions:
- This protocol facilitates the study of Leishmania uptake mechanisms by phagocytic cells.
- Understanding these pathways can lead to the discovery of new therapeutic targets for leishmaniasis.
- The described method provides a foundation for advancing leishmaniasis research and treatment development.
Abstract:
Leishmaniasis, a neglected tropical disease, is caused by the intracellular protozoan parasite Leishmania. Upon its transmission through a sandfly bite, Leishmania binds and enters host phagocytic cells, ultimately resulting in a cutaneous or visceral form of the disease. The limited therapeutics available for leishmaniasis, in combination with this parasite's techniques to evade the host immune system, call for exploring various methods to target this infection. To this end, our laboratory has been characterizing how Leishmania is internalized by phagocytic cells through the activation of multiple host cell signaling pathways. This protocol, which we use routinely for our experiments, delineates how to infect mammalian macrophages with either promastigote or amastigote forms of the Leishmania parasite. Subsequently, the number of intracellular parasites, external parasites, and macrophages can be quantified using immunofluorescence microscopy and semi-automated analysis protocols. Studying the pathways that underlie Leishmania uptake by phagocytes will not only improve our understanding of these host-pathogen interactions but may also provide a foundation for discovering additional treatments for leishmaniasis. Key features • This protocol visualizes and quantifies multiple intracellular forms of Leishmania. • It offers flexibility at various points for researchers to introduce modifications according to their study needs.
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