Lu H G, Zhong L, Chang K P, Docampo R
Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802, USA.
J Biol Chem. 1997 Apr 4;272(14):9464-73. doi: 10.1074/jbc.272.14.9464.
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.
墨西哥利什曼原虫亚马逊亚种前鞭毛体或无鞭毛体的强毒株和无毒株用荧光试剂fura 2/AM负载，以测量细胞内游离钙（[Ca2+]i）。当在名义上无细胞外Ca2+的情况下用钙离子载体离子霉素处理细胞时，[Ca2+]i增加，添加NH4Cl、尼日利亚菌素或液泡H+-ATP酶抑制剂巴弗洛霉素A1后进一步升高。当添加顺序颠倒时也获得了类似结果。考虑到离子霉素可释放的Ca2+池和离子霉素加NH4Cl可释放的Ca2+池的相对重要性，很明显，储存在墨西哥利什曼原虫亚马逊亚种前鞭毛体和无鞭毛体中的大量Ca2+存在于富含Ca2+的酸性区室（酸性钙小体）中。结果表明，与强毒前鞭毛体或两个阶段的无毒细胞相比，无毒无鞭毛体细胞内储存的可释放Ca2+更多。这种较高量的可释放Ca2+与巨噬细胞入侵期间强毒无鞭毛体中Ca2+信号的存在相关。通过用细胞内Ca2+螯合剂（1,2-双（邻氨基苯氧基）乙烷-N,N,N',N'-四乙酸/AM）和喹啉2/AM）预加载寄生虫可降低Ca2+信号和入侵，但用非Ca2+螯合类似物（N-（2-甲氧基苯基）亚氨基乙酸/AM）则不能。克隆并测序了编码细胞器型Ca2+-ATP酶的基因，发现与所有其他形式相比，该基因在强毒无鞭毛体中过表达。总之，这些结果表明Ca2+-ATP酶的表达、细胞内Ca2+池含量和信号传导与毒力之间存在显著联系。
