Impact of Reactive Oxygen Species produced by their sand fly vector on Leishmania sexual hybrid formation

Project Information

SF-Leishyb

Grant agreement ID: 101065432

DOI

10.3030/101065432

Project closed

EC signature date 13 July 2022

Start date 1 September 2022

End date 31 August 2024

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 211 754,88

Coordinated by

INSTITUT PASTEUR
France

Periodic Reporting for period 1 - SF-Leishyb (Impact of Reactive Oxygen Species produced by their sand fly vector on Leishmania sexual hybrid formation)

Reporting period: 2022-09-01 to 2024-08-31

Summary of the context and overall objectives of the project

Leishmania parasites are the causative agents of leishmaniasis, a disease characterized by a wide spectrum of symptoms ranging from cutaneous lesions to fatal visceral damages. This large diversity of clinical outcomes is due to the high genetic diversity of the parasite strains. Leishmania typically reproduce by clonal division but on rare occasions they can also undergo a cryptic sexual cycle, resulting in the production of hybrid progeny. This sexual mating is believed to be a significant source of genetic diversity in Leishmania, and can lead to changes in tissue tropism, pathology, or even drug resistance. Leishmania hybrids can be generated experimentally, in vivo by co-infecting Phlebotomine sand flies (the vector insects harboring Leishmania) or in in vitro axenic cultures. However, the frequency of hybrids generated in vivo is notably much higher than in vitro, despite the ability to culture large quantities of parasites, indicating that the gut environment is particularly conducive to Leishmania sexual mating. The exact mechanisms of Leishmania sexual reproduction and the factors that make the vector’s gut favorable for this process remain unknown and are the focus of this project.
I previously showed that exposing parental parasite cultures to Reactive Oxygen Species (ROS) increases hybrid production in vitro, which led me to hypothesize that ROS, present in the sand fly gut, could be a key factor in promoting Leishmania sexual reproduction. The project SF-Leishyb aimed to explore the mechanisms of Leishmania hybrid production and the investigate the impact of ROS present in the sand fly gut on the engagement of the parasites in a sexual reproduction path. The project presented two objectives: 1) to decipher the impact of the ROS present in the sand fly gut on Leishmania sexual reproduction in vivo by modify the ROS gut levels through food supplementation and/or sand fly genome editing; and 2) to uncover the role of Leishmania candidate genes including oxidant detoxification enzymes and DNA repair factors on parasite sexual reproduction through parasite genome editing and experimental hybrid production.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Objective 1: We performed experimental infections of Lutzomyia longipalpis sand flies with Leishmania tropica parasites and modified ROS levels in the sand fly gut through the supplementation of antioxidants (ascorbic acid) or ROS (hydrogen peroxide) in their diet. We observed that the addition of ascorbic acid leads to an increased quantity of parasites per gut, whereas on the contrary hydrogen peroxide addition causes a reduction of the number of parasites per gut. This result shows that ROS in the sand fly gut affects the ability of the parasites to colonize the gut. As we couldn’t establish a mutant sand fly line with modified ROS levels and test if a modification of the gut ROS levels impacts the Leishmania ability to produce hybrids, we instead focused our efforts on in vitro hybridization experiments. We tested various conditions and strains combinations to improve the efficiency of our assays and demonstrated that, unexpectedly, the strain Leishmania donovani Ld1S produces much more hybrids than any other Leishmania strain tested so far. This is an exciting result because the low frequency of hybridization events has historically been a major limitation to assess the mechanisms of Leishmania sexual reproduction. Comparing this strain with other, less mating-competent Leishmania strains could allow us to uncover determinants of sexual compatibility and reproduction. We then analysed the genomic inheritance patterns of hybrids resulting from the Ld1S strain self-hybridization.

Objective 2: we performed genome editing on two parental strains of Leishmania tropica and have generated several Leishmania mutant strains targeting genes of interest related to hybridization and stress response. We then did in vitro and in vivo hybridization assays with some of the genome-edited strains and could observe that null mutant strains for the gene Gex1 are completely unable to produce hybrids in vitro. We are now testing if the mutant strains for Gex1 are also unable to produce hybrids in the sand fly gut – which our preliminary experimental infections seem to indicate. Gex1 is the first factor reported as essential for sexual reproduction in Leishmania, and we are currently investigating its mode of action.
Additionally, we established a scientific collaboration with the laboratory of Domenico Otranto, at Bari University (Italy), in order to test if two very distinct Leishmania strains circulating in the same geographical area and recently shown to be able to infect the same sand fly specie - namely L. tarentolae and L. infantum - were able to hybridize and then to characterize the phenotype of eventual hybrids. We successfully produced 9 L. infantum/L. infantum hybrids and a single L. tarentolae/L. infantum hybrid, which demonstrated the possibility of sexual reproduction between these very distant strains. We then analyzed the genomic content of this unusual hybrid compared to its parents, its transcriptome and the immune reaction it induces in dog and mouse macrophages. We demonstrated that the inheritance pattern of the parental genomes is complex and varies from one chromosome to another. Despise its complex genomic features, the hybrid elicits the same cytokine production from dog macrophages as its parents.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The research performed for the SF-Leishyb project demonstrated that reactive oxygen species (ROS) in Lutzomyia longipalpis sand fly guts affect Leishmania tropica's ability to establish infection. In addition, we identified the Leishmania gene Gex1 as essential for the hybridization process: by performing experimental hybridization assays, we showed that Gex1 is essential for hybridization and is needed in only one of the two parental cells. This is an important result, as no other gene has been described as completely essential for Leishmania hybridization in the literature so far. Sexual reproduction mechanisms in Leishmania and other trypanosomatid parasites remain poorly understood, despite hybridization being a key driver of genomic plasticity. This plasticity influences important parasite traits like tissue tropism, virulence, and drug resistance. The study's findings contribute significantly to this emerging field. Our ongoing research is focused on understanding Gex1's mode of action and exploring how ROS levels in the gut affect its expression.
More over, I could evidence the high capability of the strain Leishmania donovani Ld1S to engage in sexual reproduction. This result – and the characterization of the resulting hybrids - is an important milestone in the experimental study of Leishmania reproduction, that has been historically limited by the rarity of hybridization events. Finally, the collaboration we set up with our colleagues of Bari University led us to produce a very unusual hybrid as well as key findings regarding its genomic composition, genic expression and phenotype of Leishmania hybrids.

Together, our results provide significant new insights into the mechanisms of sexual reproduction in Leishmania parasites, a process that has long been elusive, despise its relevance in the phenotypic manifestations of these pathogens.

graphical abstract of the project