Identification of a novel bicarbonate transporter critical for Leishmania virulence

Abstract

Leishmania parasites survive and proliferate within the acidic phagolysosomes of host macrophages while maintaining a near-neutral cytosolic pH, a process essential for intracellular survival and virulence. Although our prior work implicated a bicarbonate-based buffering mechanism, the absence of any annotated bicarbonate transporter in the parasite genome has hindered its identification. Using an isotope ratio mass spectrometry-based assay, we first confirmed the presence of a functional bicarbonate transport system in the parasite. Guided by this, a noncanonical homology search identified an uncharacterized protein, hereafter designated LmSLC26A, with similarity to metazoan SLC26 family of multi-anion transporters. LmSLC26A localized to the parasite plasma membrane and, under acidic conditions, was found to associate with the membrane carbonic anhydrase LmCA2. Disruption of a single LmSLC26A allele significantly reduced bicarbonate uptake, leading to intracellular acidification that was reversible upon exogenous bicarbonate supplementation. LmSLC26A-deficient parasites exhibited sluggish growth, increased apoptosis, reduced production of virulence associated cAMP and impaired exosome release. Consequently, these mutant parasites showed diminished survival within host macrophages and markedly attenuated virulence in mice. Intriguingly, LmSLC26A harbours a unique histidine ecto-phosphatase domain, revealing an unprecedented functional coupling between bicarbonate transport and enzymatic activity. Together, these findings establish LmSLC26A as the first identified bicarbonate transporter not only in Leishmania but also across the broader trypanosomatid and protozoan lineages, and define bicarbonate transport as a critical determinant of pH homeostasis and pathogenicity in the parasite. Significance StatementLeishmania parasites proliferate within the acidic phagolysosomes of host macrophages, yet the mechanism by which they maintain intracellular pH homeostasis has remained incompletely understood. Here, we identify LmSLC26A as the first bicarbonate transporter discovered in Leishmania and, more broadly, in protozoan parasites and demonstrate its essential role in bicarbonate uptake and maintenance of cytosolic pH homeostasis. LmSLC26A-deficient parasites exhibited impaired growth, increased apoptosis, reduced virulence-associated cAMP production and exosome release, resulting in compromised intracellular survival and attenuated virulence in mice. Intriguingly, LmSLC26A harbours a unique histidine ecto-phosphatase domain, revealing a previously unrecognized functional coupling between bicarbonate transport and enzymatic activity. Collectively, these findings establish LmSLC26A as a central regulator of parasite physiology and virulence.