title: Perforin enhances the granulysin-induced lysis of Listeria innocua in human dendritic cells.
abstract: BACKGROUND
  Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play an essential role in the host defence against intracellular pathogens such as Listeria, and Mycobacteria. The key mediator of bacteria-directed cytotoxicity is granulysin, a 9 kDa protein stored in cytolytic granules together with perforin and granzymes. Granulysin binds to cell membranes and is subsequently taken up via a lipid raft-associated mechanism. In dendritic cells (DC) granulysin is further transferred via early endosomes to L. innocua-containing phagosomes were bacteriolysis is induced. In the present study we analysed the role of perforin in granulysin-induced intracellular bacteriolysis in DC.
  
  
  RESULTS
  We found granulysin-induced lysis of intracellular Listeria significantly increased when perforin was simultaneously present. In pulse-chase experiments enhanced bacteriolysis was observed when perforin was added up to 25 minutes after loading the cells with granulysin demonstrating no ultimate need for simultaneous uptake of granulysin and perforin. The perforin concentration sufficient to enhance granulysin-induced intracellular bacteriolysis did not cause permanent membrane pores in Listeria-challenged DC as shown by dye exclusion test and LDH release. This was in contrast to non challenged DC that were more susceptible to perforin lysis. For Listeria-challenged DC, there was clear evidence for an Ca2+ influx in response to sublytic perforin demonstrating a short-lived change in the plasma membrane permeability. Perforin treatment did not affect granulysin binding, initial uptake or intracellular trafficking to early endosomes. However, enhanced colocalization of granulysin with listerial DNA in presence of perforin was found by confocal laser scanning microscopy.
  
  
  CONCLUSIONS
  The results provide evidence that perforin increases granulysin-mediated killing of intracellular Listeria by enhanced phagosome-endosome fusion triggered by a transient Ca2+ flux.
authors: Institute of Anatomy, Division of Cell Biology, University of Zurich, Zurich, Switzerland. michael.walch@anatom.unizh.ch
authors: Michael
authors: M
authors: Walch
authors: Sonja
authors: S
authors: Latinovic-Golic
authors: Ana
authors: A
authors: Velic
authors: Hanna
authors: H
authors: Sundstrom
authors: Claudia
authors: C
authors: Dumrese
authors: Carsten A
authors: CA
authors: Wagner
authors: Peter
authors: P
authors: Groscurth
authors: Urs
authors: U
authors: Ziegler
mesh_terms: D000945:Antigens, Differentiation, T-Lymphocyte / Q000494:pharmacology / Q000502:physiology*; D001433:Bacteriolysis / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D003602:Cytotoxicity, Immunologic / Q000187:drug effects / Q000502:physiology*; D003713:Dendritic Cells / Q000187:drug effects / Q000382:microbiology / Q000502:physiology*; D011992:Endosomes / Q000187:drug effects / Q000382:microbiology / Q000502:physiology; D006801:Humans; D008087:Listeria / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000494:pharmacology / Q000502:physiology*; D021962:Membrane Microdomains; D050296:Microbial Viability; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins / Q000494:pharmacology / Q000502:physiology*; D011994:Recombinant Proteins / Q000494:pharmacology
chemical_list: D000945:Antigens, Differentiation, T-Lymphocyte; C075005:GNLY protein, human; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; D054353:Perforin; D002118:Calcium
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