Calnexin, calreticulin and cytoskeleton-associated proteins modulate uptake and growth of Legionella pneumophila in Dictyostelium discoideum.
The haploid amoeba Dictyostelium discoideum is a versatile host system for studying cellular aspects of Legionella pathogenicity. Previous studies have shown that the internalization of L. pneumophila leads 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 of L. pneumophila. Confocal microscopic time series with green fluorescent protein (GFP)-tagged calnexin and calreticulin demonstrated the accumulation of both proteins in the phagocytic cup of L. pneumophila-infected host cells. In contrast to the control experiment with Escherichia coli-containing phagosomes, both proteins decorated the replicative vacuole of L. pneumophila during 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 of L. pneumophila in calnexin- and calreticulin-minus cells suggest that these factors are part of a regulatory system that leads to the specific vacuole of L. pneumophila.
Fajardo
Marcela
M
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany.
Schleicher
Michael
M
Institut für Zellbiologie, Ludwig-Maximilians-Universität, Schillerstr. 42, D-80336 München, Germany.
Noegel
Angelika
A
Zentrum Biochemie, Medizinische Fakultät der Universität zu Köln, Joseph-Stelzmann-Str. 52, D-50931 Köln, Germany.
Bozzaro
Salvatore
S
Department of Clinical and Biological Sciences, University Turin, Ospedale S. Luigi, Orbassano 10043, Italy.
Killinger
Silke
S
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany.
Heuner
Klaus
K
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany.
Hacker
Jörg
J
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany.
Steinert
Michael
M
Institut für Molekulare Infektionsbiologie, Universität Würzburg, Röntgenring 11, D-97070 Würzburg, Germany.
D000818:Animals; D019677:Artificial Gene Fusion; D002118:Calcium / Q000502:physiology; D002135:Calcium-Binding Proteins / Q000502:physiology; D037281:Calnexin / Q000235:genetics / Q000502:physiology*; D037282:Calreticulin / Q000235:genetics / Q000502:physiology*; D015169:Colony Count, Microbial; D003593:Cytoplasm / Q000737:chemistry; D003599:Cytoskeleton / Q000502:physiology*; D004023:Dictyostelium / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000382:microbiology*; D017353:Gene Deletion; D017930:Genes, Reporter; D016952:Legionella pneumophila / Q000254:growth & development* / Q000378:metabolism / Q000472:pathogenicity; D018613:Microscopy, Confocal; D009154:Mutation; D010587:Phagocytosis / Q000187:drug effects; D015800:Protozoan Proteins / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014617:Vacuoles / Q000737:chemistry / Q000382:microbiology / Q000648:ultrastructure
D002135:Calcium-Binding Proteins; D037282:Calreticulin; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D037281:Calnexin; D002118:Calcium