Localization of intracellular Ca2+ stores in HeLa cells during infection with Chlamydia trachomatis

Chlamydia trachomatis elementary bodies (EBs) enter epithelial cells within membrane-bound endosomes that aggregate with each other in a calcium-regul ated process, but avoid fusion with lysosomes, Annexin III but not I transl ocates to chlamydial aggregates and inclusions. In this study, we localize the intracellular Ca2+ stores during the course of infection by analyzing t he distribution of three intracellular Ca2+ store proteins: calreticulin, t ype-1 inositol-1,4,5-trisphosphate receptor (IP3-R), and Sarcoplasmic/Endop lasmic Reticulum Ca2+ ATPase type 2 (SERCA2) in HeLa cells infected with C. trachomatis serovar L2, In uninfected cells, immunofluorescence staining o f the proteins showed a fine granular distributed pattern for all three pro teins. After infection with C. trachomatis, calreticulin was found at the p eriphery of chlamydial aggregates and inclusions from 3 to 48 hours post-in fection. In infected cells, SERCA2 was intimately associated with chlamydia l inclusions after 3 and 24 hours, but not after 48 hours. Moreover, IP3-R was translocated to and colocalized with EB aggregates and chlamydial inclu sions and had a distribution very similar to that of SERCA2, After 24 hours incubation with chlamydiae, there was a local accumulation o f [Ca2+](i) (105+/-17 nM) in the proximity of chlamydial inclusions, compar ed to 50+/-13 nM in other parts of the cell cytoplasm. In the absence of ex tracellular Ca2+, this local accumulation of Ca2+ increased to 295+/-50 nM after adding 50 mu M ATP, and to a similar extent after adding 100 nM thaps igargin (Tg), These data indicate that during infection of HeLa cells with chlamydiae, intracellular Ca2+ stores are redistributed, causing local accu mulation of Ca2+ in the vicinity of chlamydial inclusions, These changes ma y trigger the association of certain proteins such as annexins with chlamyd ia-containing vesicles, and thereby regulation of membrane-membrane interac tion during endosome aggregation and inclusion formation.