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Infection and Immunity, September 2009, p. 3611-3625, Vol. 77, No. 9
0019-9567/09/$08.00+0     doi:10.1128/IAI.01478-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Staphylococcal Alpha-Toxin Is Not Sufficient To Mediate Escape from Phagolysosomes in Upper-Airway Epithelial Cells {triangledown} ,{dagger}

Bernd Giese,1 Silvia Dittmann,1 Kerstin Paprotka,1 Katja Levin,1 Annett Weltrowski,1 Diana Biehler,1 Thiên-Trí Lâm,2 Bhanu Sinha,2 and Martin J. Fraunholz1*

Competence Center for Functional Genomics, Ernst Moritz Arndt University, F. L. Jahnstrasse 15, D-17487 Greifswald, Germany,1 Institute of Hygiene and Microbiology, University of Würzburg, Josef Schneider Str. 2, Bldg. E1, D-97080 Würzburg, Germany2

Received 3 December 2008/ Returned for modification 11 May 2009/ Accepted 8 June 2009

Intracellular Staphylococcus aureus has been implicated in the establishment of chronic infections. It is therefore imperative to understand by what means S. aureus is able to survive within cells. Here we use two expression systems with a fluorescent readout to assay alpha-toxin expression and function within phagolysosomes of infected upper-airway epithelial cells: avirulent Staphylococcus carnosus TM300 and phenotypically alpha-toxin-negative S. aureus laboratory strains. Data from CFU recovery assays suggest that the presence of alpha-toxin is not beneficial for the intracellular survival of recombinant Staphylococcus strains. This finding was corroborated by immunofluorescence studies: whereas S. carnosus and S. aureus are able to deliver S. aureus alpha-toxin to lumina of host cell phagolysosomes, the membrane integrity of these organelles was not affected. Alpha-toxin-expressing strains were detected exclusively within lysosome-associated membrane protein 1 (LAMP1)-yellow fluorescent protein (YFP)-positive vesicles. Measurements of intraphagosomal pH illustrated that all infected phagolysosomes acidified regardless of alpha-toxin expression. In contrast, S. aureus expressing Listeria monocytogenes listeriolysin O leads to the breakdown of the phagolysosomal membrane, as indicated by staphylococci that are not associated with LAMP1-YFP-decorated vesicles and that do not reside within an acidic cellular environment. Thus, our results suggest that staphylococcal alpha-toxin is not sufficient to mediate phagolysosomal escape in upper-airway epithelial cells.

* Corresponding author. Mailing address: FunGene, Competence Center for Functional Genomics, E. M. Arndt University, F. L. Jahnstrasse 15, D-17487 Greifswald, Germany. Phone: 49 (0)3834 864072. Fax: 49 (0)3834 864073. E-mail: Martin.Fraunholz{at}uni-greifswald.de

{triangledown} Published ahead of print on 29 June 2009.

{dagger} Supplemental material for this article may be found at http://iai.asm.org/.

Editor: J. B. Bliska

Infection and Immunity, September 2009, p. 3611-3625, Vol. 77, No. 9
0019-9567/09/$08.00+0     doi:10.1128/IAI.01478-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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