Periodic Reporting for period 1 - LeishMOM (Inadequate migration of Leishmania-infected macrophages – the driver of parasite dissemination?)
Periodo di rendicontazione: 2023-09-01 al 2025-08-31
Leishmaniasis is a major neglected tropical disease, with over one million new cases and approximately 20,000 deaths reported annually. Transmitted through the bite of infected sand flies, Leishmania parasites invade macrophages, subverting these immune cells to survive and replicate. Depending on the infecting species, disease manifestations range from localised cutaneous lesions (cutaneous leishmaniasis [CL]) to fatal visceral infections (visceral leishmaniasis [VL]). The difference between a mild and a life-threatening form of disease is largely determined by how the parasite disseminates within the host, yet the mechanisms driving this dissemination remain poorly defined.
Previous work suggests that Leishmania species can manipulate macrophage migration in a species-dependent manner. Understanding how Leishmania species hijack host migration pathways is essential for identifying new therapeutic strategies that target host-pathogen interactions.
This project set out to test the hypothesis that Leishmania species modulate macrophage migration in a species-specific manner. The work was structured around two main objectives:
1. To compare the spatiotemporal migration behaviour of macrophages infected with species causing cutaneous and visceral leishmaniasis.
2. To identify host macrophage genes and molecular pathways underlying these migration phenotypes using CRISPR knockout and drug-inhibitor screens.
Previous work suggests that Leishmania species can manipulate macrophage migration in a species-dependent manner. Understanding how Leishmania species hijack host migration pathways is essential for identifying new therapeutic strategies that target host-pathogen interactions.
This project set out to test the hypothesis that Leishmania species modulate macrophage migration in a species-specific manner. The work was structured around two main objectives:
1. To compare the spatiotemporal migration behaviour of macrophages infected with species causing cutaneous and visceral leishmaniasis.
2. To identify host macrophage genes and molecular pathways underlying these migration phenotypes using CRISPR knockout and drug-inhibitor screens.
Objective 1:
To dissect the migration dynamics of Leishmania-infected macrophages, we employed 2D motility assays to investigate the migration patterns of THP-1- and BLaER1-derived macrophages infected with different Leishmania species. Using a low infection ratio to minimise external influences on macrophage behaviour, we conducted detailed tracking analyses across 360 individual macrophage infection experiments. Our findings reveal that VL-causing species, L. donovani and L. infantum, enhance macrophage motility, whereas the CL-causing species L. mexicana suppresses it. These motility differences are associated with parasite-specific alterations in macrophage morphology and actin cytoskeletal dynamics. L. donovani-infected macrophages displayed reduced circularity, increased actin-based protrusions, and higher total actin content. Conversely, L. mexicana-infected macrophages were more rounded and exhibited diminished actin signals. Importantly, these parasite-driven effects were distinct from those observed in bead-bearing macrophages. These species-specific manipulations were consistent across various experimental conditions, including temperatures mimicking skin and organ environments, differing parasite loads, and varying in vitro passage numbers.
In addition, we tracked cells migrating through extracellular matrix (ECM) gels of different densities and demonstrated that Leishmania parasites themselves do not exhibit sufficient motility in collagen type I matrices to contribute meaningfully to parasite dissemination, whereas macrophages migrate efficiently in these 3D environments. However, tracking of Leishmania-infected and bead-bearing macrophages in these 3D matrices is still ongoing and has proven more challenging than originally anticipated. We also encountered difficulties when attempting to track macrophages in artificial human skin models. Here macrophages showed low viability and almost no migration capacity within these models, and therefore, these skin models were not used further in this project.
Objective 2:
To identify host macrophage genes and molecular pathways underlying these migration phenotypes, we established a lentiviral-delivered CRISPR/Cas12a gene-editing system in THP-1 cells suitable for multiplexed CRISPR screening. We successfully created several proof-of-principle knockouts, including paxillin and MMP9, and are currently analysing their phenotypes during Leishmania infection. Efforts to upscale the workflow and combine CRISPR screening with transwell assays to identify genetic host factors influencing migration in THP-1-derived macrophages manipulated by Leishmania in a species-dependent manner are ongoing.
In addition, we conducted pharmacological inhibitor screens to investigate the host pathways modulated by Leishmania species during macrophage migration. Two inhibitors were selected: CK-666, which blocks Arp2/3-mediated actin polymerisation, and (S)-nitro-Blebbistatin, which inhibits non-muscle myosin II ATPase activity and thereby affects the Rho/ROCK pathway. Migration assays using THP-1 and BLaER-1 derived macrophages infected with L. donovani and L. mexicana revealed distinct effects. CK-666 treatment reduced overall macrophage motility, and L. donovani infection did not overcome this inhibition, indicating that parasite-induced motility enhancement depends on Arp2/3-driven actin polymerisation and complements our findings that Leishmania manipulates the actin content of the host cell. In contrast, Blebbistatin reduced motility in bead-bearing macrophages, but L. donovani-infected cells maintained or slightly increased migration, suggesting that parasite-mediated motility changes are largely independent of myosin II activity.
Together, our results provide new mechanistic insights into how Leishmania species-specifically manipulates macrophage migration and host cytoskeletal pathways to promote dissemination.
To dissect the migration dynamics of Leishmania-infected macrophages, we employed 2D motility assays to investigate the migration patterns of THP-1- and BLaER1-derived macrophages infected with different Leishmania species. Using a low infection ratio to minimise external influences on macrophage behaviour, we conducted detailed tracking analyses across 360 individual macrophage infection experiments. Our findings reveal that VL-causing species, L. donovani and L. infantum, enhance macrophage motility, whereas the CL-causing species L. mexicana suppresses it. These motility differences are associated with parasite-specific alterations in macrophage morphology and actin cytoskeletal dynamics. L. donovani-infected macrophages displayed reduced circularity, increased actin-based protrusions, and higher total actin content. Conversely, L. mexicana-infected macrophages were more rounded and exhibited diminished actin signals. Importantly, these parasite-driven effects were distinct from those observed in bead-bearing macrophages. These species-specific manipulations were consistent across various experimental conditions, including temperatures mimicking skin and organ environments, differing parasite loads, and varying in vitro passage numbers.
In addition, we tracked cells migrating through extracellular matrix (ECM) gels of different densities and demonstrated that Leishmania parasites themselves do not exhibit sufficient motility in collagen type I matrices to contribute meaningfully to parasite dissemination, whereas macrophages migrate efficiently in these 3D environments. However, tracking of Leishmania-infected and bead-bearing macrophages in these 3D matrices is still ongoing and has proven more challenging than originally anticipated. We also encountered difficulties when attempting to track macrophages in artificial human skin models. Here macrophages showed low viability and almost no migration capacity within these models, and therefore, these skin models were not used further in this project.
Objective 2:
To identify host macrophage genes and molecular pathways underlying these migration phenotypes, we established a lentiviral-delivered CRISPR/Cas12a gene-editing system in THP-1 cells suitable for multiplexed CRISPR screening. We successfully created several proof-of-principle knockouts, including paxillin and MMP9, and are currently analysing their phenotypes during Leishmania infection. Efforts to upscale the workflow and combine CRISPR screening with transwell assays to identify genetic host factors influencing migration in THP-1-derived macrophages manipulated by Leishmania in a species-dependent manner are ongoing.
In addition, we conducted pharmacological inhibitor screens to investigate the host pathways modulated by Leishmania species during macrophage migration. Two inhibitors were selected: CK-666, which blocks Arp2/3-mediated actin polymerisation, and (S)-nitro-Blebbistatin, which inhibits non-muscle myosin II ATPase activity and thereby affects the Rho/ROCK pathway. Migration assays using THP-1 and BLaER-1 derived macrophages infected with L. donovani and L. mexicana revealed distinct effects. CK-666 treatment reduced overall macrophage motility, and L. donovani infection did not overcome this inhibition, indicating that parasite-induced motility enhancement depends on Arp2/3-driven actin polymerisation and complements our findings that Leishmania manipulates the actin content of the host cell. In contrast, Blebbistatin reduced motility in bead-bearing macrophages, but L. donovani-infected cells maintained or slightly increased migration, suggesting that parasite-mediated motility changes are largely independent of myosin II activity.
Together, our results provide new mechanistic insights into how Leishmania species-specifically manipulates macrophage migration and host cytoskeletal pathways to promote dissemination.
In addition to these results, we have also developed new genetic tools for Leishmania species, allowing the pursuit of genome-wide loss-of-function screens in Leishmania (Engstler, M. and T. Beneke, eLife 2023; Herrmann May et al., eLife 2025; Arias-del-Angel et al., BioRxiv 2025). Our use of a CRISPR/Cas9 cytosine base editor (CBE) facilitates the introduction of premature STOP codons or other functional mutations into any gene of interest, generating non-clonal mutant populations. Notably, this method allows targeting of both coding and non-coding genes and is particularly suited for the repetitive Leishmania genome. Coupled with a Cas12a-mediated system for integrating CBE sgRNA libraries into a genomic safe-harbour locus, our system now enables, for the first time, genome-wide high-throughput loss-of-function screening in Leishmania.
In the future, the fellow aims to identify genetic Leishmania factors that contribute to the observed modifications of macrophage migration.
In the future, the fellow aims to identify genetic Leishmania factors that contribute to the observed modifications of macrophage migration.