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 ([Ca2+]i). When the cells were treated with the calcium ionophore ionomycin in the nominal absence of extracellular Ca2+, there was an increase of [Ca2+]i that was further elevated by addition of either NH4Cl, nigericin, or the vacuolar H+-ATPase inhibitor bafilomycin A1. 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 NH4Cl-releasable Ca2+ pools, it is apparent that a significant amount of the Ca2+ stored in L. mexicana amazonensis promastigotes and amastigotes is present in an acidic compartment rich in Ca2+ (acidocalcisome). Results indicated that more releasable Ca2+ is stored intracellularly in virulent amastigotes than in virulent promastigotes or avirulent cells of both stages. This higher amount of releasable Ca2+ was correlated with the presence of Ca2+ signals in the virulent amastigotes during invasion of macrophages. Ca2+ signals and invasion were reduced by preloading the parasites with intracellular Ca2+ chelators (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM) and quin 2/AM) but not by a non-Ca2+-chelating analog (N-(2-methoxyphenyl)imidoacetic acid/AM). The gene encoding an organelle-type Ca2+-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 Ca2+-ATPase, intracellular Ca2+ pool content and signaling, and virulence.
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Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA.
Zhong
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Chang
K P
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Docampo
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D000595:Amino Acid Sequence; D000634:Aminoquinolines / Q000494:pharmacology; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D007202:Indicators and Reagents / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007894:Leishmania mexicana / Q000201:enzymology / Q000472:pathogenicity*; D018942:Macrolides; D008264:Macrophages / Q000469:parasitology; D008647:Mesocricetus; D008969:Molecular Sequence Data; D009550:Nigericin / Q000494:pharmacology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D015398:Signal Transduction
D000634:Aminoquinolines; D000900:Anti-Bacterial Agents; D007202:Indicators and Reagents; D007476:Ionophores; D018942:Macrolides; D010975:Platelet Aggregation Inhibitors; D000643:Ammonium Chloride; D004533:Egtazic Acid; D015759:Ionomycin; C040929:bafilomycin A1; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; C035739:Quin2; D009550:Nigericin; D002118:Calcium