Interplay between a cytosolic and a cell surface carbonic anhydrase in pH homeostasis and acid tolerance of Leishmania

Abstract

Leishmania parasites have evolved to endure the acidic phagolysosomal environment within host macrophages. How Leishmania cells maintain near-neutral intracellular pH and proliferate in such a proton-rich mileu remains poorly understood. We report here that, in order to thrive in acidic conditions, Leishmania major relies on a cytosolic and a cell surface carbonic anhydrase, LmCA1 and LmCA2, respectively. Upon exposure to acidic medium, the intracellular pH of the LmCA1 +/− , LmCA2 +/− and LmCA1 +/− :LmCA2 +/− mutant strains dropped by varying extents that led to cell cycle delay, growth retardation and morphological abnormalities. Intracellular acidosis and growth defects of the mutant strains could be reverted by genetic complementation or supplementation with bicarbonate. When J774A.1 macrophages were infected with the mutant strains, they exhibited much lower intracellular parasite burdens than their wild-type counterparts. However, these differences in intracellular parasite burden between the wild-type and mutant strains were abrogated if, before infection, the macrophages were treated with chloroquine to alkalize their phagolysosomes. Taken together, our results demonstrate that haploinsufficiency of LmCA1 and/or LmCA2 renders the parasite acid-susceptible, thereby unravelling a carbonic anhydrase-mediated pH homeostatic circuit in Leishmania cells.

Potential Antileishmanial Activity of a Triazole‐Based Hybrid Peptide against Leishmania major

“…To investigate whether peptide 1 have any inhibitory effect on mammalian cell or not cell viability test was performed for peptide 1 on J774 A.1 murine macrophage cell line. J774 A.1 murine macrophage cell line were cultured as described previously by Pal et al . Macrophage (J774 A.1) cells were grown in presence of different concentrations of the compound with a cell density of 1 X 10 5 cells/ml.…”

“…Hence, we conclude that the peptide 1 does not have any “cidal” effects to the Leishmania cells rather we can call the peptide 1 as “static” agent against Leishmania . Morphological abnormality of Leishmania (especially shorter size and rounded shape) is observed during cellular stress . Thus peptide 1 treated cells due to inhibition of metabolic processes experienced a significant cellular stress which may not kill the parasite but halted the normal growth in vitro and resulted in morphological abnormality.…”

Lathosterol oxidase (sterol C5-desaturase) deletion confers resistance to amphotericin B and sensitivity to acidic stress inLeishmania major

“…4C) suggests that their plasma membrane is less stable, thus affecting their ability to control intracellular pH. Besides plasma membrane, certain intracellular organelles and proteins are essential for intracellular pH homeostasis as well (38). Acidocalcisomes are electron-dense acidic organelles rich in divalent cations and polyphosphate first identified in trypanosomatids (39).…”

Leishmaniainfection triggers hepcidin-mediated proteasomal degradation of Nramp1 resulting in increased phagolysosomal iron availability

“…J774A.1 cells (murine macrophage cell line obtained from National Center for Cell Sciences, Pune) were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) pH 7.4 supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat-inactivated FBS at 37°C in a humidified atmosphere containing 5% CO2. Cell number was quantified using a hemocytometer (10).…”

“…Infection of J774A.1 or primary peritoneal macrophages with L. major promastigotes was performed as described by us previously (10). Briefly, the macrophages were activated with 100 ng/ml lipopolysaccharide (LPS) for 6 hours.…”