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J. Biol. Chem., Vol. 276, Issue 25, 23056-23064, June 22, 2001

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A Spirochete Surface Protein Uncouples Store-operated Calcium Channels in Fibroblasts
A NOVEL CYTOTOXIC MECHANISM^*

Qin Wang, Kevin S. Ko^§¶, András Kapus, Christopher A. G. McCulloch^§, and Richard P. Ellen^**

From the  Dental Research Institute, the ^§ Canadian Institutes of Health Research Group in Periodontal Physiology, Faculty of Dentistry, and the  Department of Surgery, University of Toronto and the Division of Surgery, Toronto General Hospital, Toronto, Ontario M5G 1G6, Canada

The cytotoxicity of infectious agents can be mediated by disruption of calcium signaling in target cells. Outer membrane proteins of the spirochete Treponema denticola, a periodontal pathogen, inhibit agonist-induced Ca²⁺ release from internal stores in gingival fibroblasts, but the mechanism is not defined. We determined here that the major surface protein (Msp) of T. denticola perturbs calcium signaling in human fibroblasts by uncoupling store-operated channels. Msp localized in complexes on the cell surface. Ratio fluorimetry showed that in cells loaded with fura-2 or fura-C18, Msp induced cytoplasmic and near-plasma membrane Ca²⁺ transients, respectively. Increased conductance was confirmed by fluorescence quenching of fura-2-loaded cells with Mn²⁺ after Msp treatment. Calcium entry was blocked with anti-Msp antibodies and inhibited by chelating external Ca²⁺ with EGTA. Msp pretreatment reduced the amplitude of [Ca²⁺]_i transients upon challenge with ATP or thapsigargin. In experiments using cells loaded with mag-fura-2 to report endoplasmic reticulum Ca²⁺, Msp reduced Ca²⁺ efflux from endoplasmic reticulum stores when ATP was used as an agonist. Msp alone did not induce Ca²⁺ release from these stores. Msp inhibited store-operated influx of extracellular calcium following intracellular Ca²⁺ depletion by thapsigargin and also promoted the assembly of subcortical actin filaments. This actin assembly was blocked by chelating intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester. The reduced amplitude of agonist-induced transients and inhibition of store-operated Ca²⁺ entry due to Msp were reversed by latrunculin B, an inhibitor of actin filament assembly. Thus, Msp retards Ca²⁺ release from endoplasmic reticulum stores, and it inhibits subsequent Ca²⁺ influx by uncoupling store-operated channels. Actin filament rearrangement coincident with conformational uncoupling of store-operated calcium fluxes is a novel mechanism by which surface proteins and toxins of pathogenic microorganisms may damage host cells.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) U66256.

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