Calnexin, calreticulin and cytoskeleton-associated proteins modulate uptake and growth of Legionella pneumophila in Dictyostelium discoideum

Abstract

The haploid amoebaDictyostelium discoideumis a versatile host system for studying cellular aspects ofLegionellapathogenicity. Previous studies have shown that the internalization ofL. pneumophilaleads to an endoplasmic reticulum (ER)-derived organelle that supports intracellular replication of the bacteria. In this study a roadmap of host-cell factors involved in this process was developed. Phagocytosis assays with specific cellular inhibitors and the effects of well defined host-cell mutants revealed that cytoplasmic calcium levels, cytoskeleton-associated proteins and the calcium-binding proteins of the ER, calreticulin and calnexin, specifically influence the uptake and intracellular growth ofL. pneumophila. Confocal microscopic time series with green fluorescent protein (GFP)-tagged calnexin and calreticulin demonstrated the accumulation of both proteins in the phagocytic cup ofL. pneumophila-infected host cells. In contrast to the control experiment withEscherichia coli-containing phagosomes, both proteins decorated the replicative vacuole ofL. pneumophiladuring the entire growth phase of the bacteria. The cumulative effects of cytosolic calcium levels, the spatial distribution of calnexin and calreticulin, and the defective invasion and replication ofL. pneumophilain calnexin- and calreticulin-minus cells suggest that these factors are part of a regulatory system that leads to the specific vacuole ofL. pneumophila.