Discovery of Leishmania Bicarbonate Transporter Essential for Parasite Virulence

Seth, Arunava (2025) Discovery of Leishmania Bicarbonate Transporter Essential for Parasite Virulence. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Arunava Seth (16IP009)) 16IP009.pdf - Submitted Version Restricted to Repository staff only Download (5MB)

Abstract

Leishmania are intracellular protozoan parasites that cause leishmaniasis. They reside within acidic phagolysosomes of host macrophages. How Leishmania has adapted itself to survive in this harsh acidic environment remains largely unknown. Previously, our lab identified the interplay between two carbonic anhydrases in the intracellular pH regulation of Leishmania and predicted the involvement of a ‘hypothetical’ bicarbonate transporter. Here, we ask the question: Does that hypothetical transporter at all exist? What is its probable function in the parasite? Despite the Leishmania genome database being devoid of any ‘annotated’ bicarbonate transporter in Leishmania, our experiment using a novel ¹³C-labeled bicarbonate (H¹³CO₃⁻) isotope ratio mass spectrometry assay establishes the definite existence of such a transporter in the parasite. However, we identified a candidate gene that has a strong similarity to the human SLC26 multi-anion transporter family, naming it LmSLC26A. We validated the expression of LmSLC26A in Leishmania across both stages of the parasite. Additionally, we found it to be localized in the plasma membrane in both stages. Using the CRISPR-Cas9 genome editing technique, we generated a LmSLC26A⁺/⁻ (heterozygous mutant) Leishmania strain. This strain showed significantly less bicarbonate transport activity as determined using stable isotope bicarbonate uptake assay, thus conclusively proving that LmSLC26A is a bicarbonate transporter. The LmSLC26A⁺/⁻ strain exhibited several abnormal phenotypes, such as intracellular acidosis, sluggish growth, and increased apoptosis, all of which could be rescued by exogenous bicarbonate. Additionally, we observe reduced levels of cAMP, changes in actin distribution, and reduced mitochondrial mass, indicating global changes due to the depletion of intracellular bicarbonate. The LmSLC26A⁺/⁻ strain was also found to be significantly less virulent in the in vitro macrophage infection model as well as in infected BALB/c mice. These observations demonstrate that the Leishmania bicarbonate transporter is crucial for pH adaptation and virulence of the parasite. Furthermore, we found that LmSLC26A colocalizes with plasma membrane-localized LmCA2 under acidic conditions, and co-immunoprecipitation assays confirmed their interaction. Notably, LmSLC26A expression was upregulated under acidic conditions, indicating its crucial function in the parasite's adaptation to the phagolysosomal environment. In conclusion, we identified LmSLC26A as the first bicarbonate transporter in the Leishmania and Kinetoplastida orders, establishing its role in pH regulation and virulence. These findings provide new insights into Leishmania biology and present LmSLC26A as a potential target for novel anti-leishmanial therapies.

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