Intracellular Ca2+ Pool Content and Signaling and Expression of a Calcium Pump Are Linked to Virulence in Leishmania mexicana amazonesis Amastigotes

Virulent and avirulent clones of Leishmania mexicana amazonensis promastigotes or amastigotes were loaded with the fluorescent reagent fura 2/AM to measure intracellular free calcium ([Ca 2+ ] i ). When the cells were treated with the calcium ionophore ionomycin in the nominal absence of extracellular Ca 2+ , there was an increase of [Ca 2+ ] i that was further elevated by addition of either NH 4 Cl, nigericin, or the vacuolar H + -ATPase inhibitor bafilomycin A 1 . Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH 4 Cl-releasable Ca 2+ pools, it is apparent that a significant amount of the Ca 2+ stored in L. mexicana amazonensis promastigotes and amastigotes is present in an acidic compartment rich in Ca 2+ (acidocalcisome). Results indicated that more releasable Ca 2+ is stored intracellularly in virulent amastigotes than in virulent promastigotes or avirulent cells of both stages. This higher amount of releasable Ca 2+ was correlated with the presence of Ca 2+ signals in the virulent amastigotes during invasion of macrophages. Ca 2+ signals and invasion were reduced by preloading the parasites with intracellular Ca 2+ chelators (1,2-bis( o -aminophenoxy)ethane- N , N , N ′, N ′-tetraacetic acid/AM) and quin 2/AM) but not by a non-Ca 2+ -chelating analog ( N -(2-methoxyphenyl)imidoacetic acid/AM). The gene encoding an organelle-type Ca 2+ -ATPase was cloned and sequenced and found overexpressed in virulent amastigotes as compared with all other forms. Together, these results demonstrate a significant link between expression of a Ca 2+ -ATPase, intracellular Ca 2+ pool content and signaling, and virulence.